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Josém. Escribano - One of the best experts on this subject based on the ideXlab platform.
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visualization of the african swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent Protein P54 membrane Protein chimera
Virology, 2006Co-Authors: Bruno Hernaez, Josém. Escribano, Covadonga AlonsoAbstract:Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the P54 envelope Protein fused to the enhanced green fluorescent Protein (EGFP). The incorporation of the fusion Protein into the virus particle was confirmed immunologically and it was determined that P54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.
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the african swine fever virus dynein binding Protein P54 induces infected cell apoptosis
FEBS Letters, 2004Co-Authors: Bruno Hernaez, Josém. Escribano, Gema Diazgil, Monica Garciagallo, Jose I Quetglas, Ignacio Rodriguezcrespo, Linda K Dixon, Covadonga AlonsoAbstract:A specific interaction of ASFV P54 Protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural Protein P54 in apoptosis, transient expression experiments of P54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, P54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of P54. This is the first reported ASFV Protein which induces apoptosis.
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african swine fever virus Protein P54 interacts with the microtubular motor complex through direct binding to light chain dynein
Journal of Virology, 2001Co-Authors: Covadonga Alonso, Bruno Hernaez, Ignacio Rodriguezcrespo, Linda K Dixon, James E Miskin, Patricia Fernandezzapatero, Lourdes Soto, Carmen Canto, Josém. EscribanoAbstract:Dynein is a minus-end-directed microtubule-associated motor Protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus Protein P54 to the light chain of cytoplasmic dynein (LC8). Interaction of P54 and LC8 occurs both in vitro and in cells, and the two Proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of P54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural Protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.
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The structural Protein P54 is essential for African swine fever virus viability
Virus research, 1996Co-Authors: Fernando Rodriguez, Javier M Rodriguez, Victoria Ley, Paulino Gómez-puertas, Ramon Güimil García, Josém. EscribanoAbstract:Protein P54, one of the most antigenic structural African swine fever virus (ASFV) Proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the P54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the P54 gene and beta-glucuronidase selection marker under the control of P54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of P54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of P54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the P54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.
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characterization and molecular basis of heterogeneity of the african swine fever virus envelope Protein P54
Journal of Virology, 1994Co-Authors: Fernando Rodriguez, Javier M Rodriguez, Covadonga Alonso, A Eiras, C Alcaraz, Rafael J Yanez, Jose F Rodriguez, Josém. EscribanoAbstract:It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in Protein P54 (C. Alcaraz, A. Brun, F. Ruiz-Gonzalvo, and J. M. Escribano, Virus Res. 23:173-182, 1992). A recombinant bacteriophage expressing a 328-bp fragment of the P54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against P54 Protein. The sequence of this recombinant phage allowed the location of the P54 gene in the EcoRI E fragment of the ASFV genome. Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a Protein of 183 amino acids with a calculated molecular weight of 19,861. This Protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for Protein processing of several ASFV structural Proteins. In addition, two direct tandem repetitions were also found within this open reading frame. Further characterization of the transcription and gene product revealed that the P54 gene is translated from a late mRNA and the Protein is incorporated to the external membrane of the virus particle. A comparison of the nucleotide sequence of the P54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity. However, when P54 genes from viral clones generated by cell culture passage and coding for P54 Proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat. These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in P54, resulting in stepwise modifications in the molecular weight of the Protein. These duplications and deletions of a tandem repeat sequence array within a Protein coding region constitute a novel mechanism of genetic diversification in ASFV.
Covadonga Alonso - One of the best experts on this subject based on the ideXlab platform.
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visualization of the african swine fever virus infection in living cells by incorporation into the virus particle of green fluorescent Protein P54 membrane Protein chimera
Virology, 2006Co-Authors: Bruno Hernaez, Josém. Escribano, Covadonga AlonsoAbstract:Many stages of African swine fever virus infection have not yet been studied in detail. To track the behavior of African swine fever virus (ASFV) in the infected cells in real time, we produced an infectious recombinant ASFV (B54GFP-2) that expresses and incorporates into the virus particle a chimera of the P54 envelope Protein fused to the enhanced green fluorescent Protein (EGFP). The incorporation of the fusion Protein into the virus particle was confirmed immunologically and it was determined that P54-EGFP was fully functional by confirmation that the recombinant virus made normal-sized plaques and presented similar growth curves to the wild-type virus. The tagged virus was visualized as individual fluorescent particles during the first stages of infection and allowed to visualize the infection progression in living cells through the viral life cycle by confocal microscopy. In this work, diverse potential applications of B54GFP-2 to study different aspects of ASFV infection are shown. By using this recombinant virus it was possible to determine the trajectory and speed of intracellular virus movement. Additionally, we have been able to visualize for first time the ASFV factory formation dynamics and the cytophatic effect of the virus in live infected cells. Finally, we have analyzed virus progression along the infection cycle and infected cell death as time-lapse animations.
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the african swine fever virus dynein binding Protein P54 induces infected cell apoptosis
FEBS Letters, 2004Co-Authors: Bruno Hernaez, Josém. Escribano, Gema Diazgil, Monica Garciagallo, Jose I Quetglas, Ignacio Rodriguezcrespo, Linda K Dixon, Covadonga AlonsoAbstract:A specific interaction of ASFV P54 Protein with 8 kDa light chain cytoplasmic dynein (DLC8) has been previously characterized and this interaction is critical during virus internalization and transport to factory sites. During early phases of infection, the virus induces the initiation of apoptosis triggering activation of caspase-9 and -3. To analyze the role of the structural Protein P54 in apoptosis, transient expression experiments of P54 in Vero cells were carried out which resulted in effector caspase-3 activation and apoptosis. Interestingly, P54 mutants, lacking the 13 aa dynein-binding motif lose caspase activation ability and pro-death function of P54. This is the first reported ASFV Protein which induces apoptosis.
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african swine fever virus Protein P54 interacts with the microtubular motor complex through direct binding to light chain dynein
Journal of Virology, 2001Co-Authors: Covadonga Alonso, Bruno Hernaez, Ignacio Rodriguezcrespo, Linda K Dixon, James E Miskin, Patricia Fernandezzapatero, Lourdes Soto, Carmen Canto, Josém. EscribanoAbstract:Dynein is a minus-end-directed microtubule-associated motor Protein involved in cargo transport in the cytoplasm. African swine fever virus (ASFV), a large DNA virus, hijacks the microtubule motor complex cellular transport machinery during virus infection of the cell through direct binding of virus Protein P54 to the light chain of cytoplasmic dynein (LC8). Interaction of P54 and LC8 occurs both in vitro and in cells, and the two Proteins colocalize at the microtubular organizing center during viral infection. p50/dynamitin, a dominant-negative inhibitor of dynein-dynactin function, impeded ASFV infection, suggesting an essential role for dynein during virus infection. A 13-amino-acid domain of P54 was sufficient for binding to LC8, an SQT motif within this domain being critical for this binding. Direct binding of a viral structural Protein to LC8, a small molecule of the dynein motor complex, could constitute a molecular mechanism for microtubule-mediated virus transport.
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the african swine fever virus Proteins P54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody mediated protective immune response
Virology, 1998Co-Authors: Paulino Gomezpuertas, Covadonga Alonso, J.m. Oviedo, Fernando Rodriguez, Alejandro Brun, J.m. EscribanoAbstract:Abstract The nature of the initial interactions of African swine fever (ASF) virus with target cells is only partially known, and to date only the ASF virus Protein p12 has been identified as a viral attachment Protein. More recently, antibodies to viral Proteins P54 and p30 have been shown to neutralize the virus, inhibiting virus binding and internalization, respectively. Therefore, we investigated the role of these Proteins in the receptor-mediated ASF virus endocytosis in swine macrophages, the natural host cells. Proteins P54 and p30, released from ASF virus particles after treatment of virions with a nonionic detergent, bound to virus-sensitive alveolar pig macrophages. Binding of these Proteins was found to be specifically inhibited by neutralizing antibodies obtained from a convalescent pig or from pigs immunized with recombinant P54 or p30 Proteins. The baculovirus-expressed Proteins P54 and p30 retained the same biological properties as the viral Proteins, since they also bound specifically to these cells, and their binding was equally inhibited by neutralizing antibodies. Binding of35S-labeled recombinant P54 and p30 Proteins to macrophages was specifically competed by an excess of unlabeled P54 and p30, respectively. However, cross-binding inhibition was not observed, suggesting the existence of two different saturable binding sites for these Proteins in the susceptible cells. In addition, Protein P54 blocked the specific binding of virus particles to the macrophage, while Protein p30 blocked virus internalization. Both Proteins independently prevented virus infection and in a dose-dependent manner, suggesting that binding interactions mediated by both Proteins are necessary to give rise to a productive infection. The relevance of blockade of virus–cell interactions mediated by P54 and p30 in the protective immune response against ASF virus was then investigated. Immunization of pigs with either recombinant P54 or p30 Proteins induced neutralizing antibodies which, as expected, inhibited virus attachment or internalization, respectively. However, immunized pigs were not protected against lethal infection and the disease course was not modified in these animals. In contrast, immunization with a combination of P54 and p30 Proteins simultaneously stimulated both virus neutralizing mechanisms and modified drastically the disease course, rendering a variable degree of protection ranging from a delay in the onset of the disease to complete protection against virus infection. In conclusion, the above results strongly suggest that Proteins P54 and p30 mediate specific interactions between ASF virus and cellular receptors and that simultaneous interference with these two interactions has a complementary effect in antibody-mediated protection.
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highly specific confirmatory western blot test for african swine fever virus antibody detection using the recombinant virus Protein P54
Journal of Virological Methods, 1995Co-Authors: Carlos Alcaraz, Covadonga Alonso, J.m. Oviedo, Fernando Rodriguez, A Eiras, M De Diego, J.m. EscribanoAbstract:A Western blot technique using a recombinant Protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA. The new confirmatory Western blot is based on the use of Protein P54, one of the most antigenic ASF virus structural Proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase. The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced Proteins ranging in molecular weight between 23 and 35 kDa. The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.
Javier M Rodriguez - One of the best experts on this subject based on the ideXlab platform.
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african swine fever virus Protein p17 is essential for the progression of viral membrane precursors toward icosahedral intermediates
Journal of Virology, 2010Co-Authors: Cristina Suarez, Javier Gutierrezberzal, Germán Andrés, Maria Salas, Javier M RodriguezAbstract:The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that Protein P54, a viral structural integral membrane Protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of Protein p17, an abundant transmembrane Protein localized at the viral internal envelope, in these processes. Using an inducible virus for this Protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyProteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that Protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this Protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.
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African Swine Fever Virus Structural Protein P54 Is Essential for the Recruitment of Envelope Precursors to Assembly Sites
Journal of virology, 2004Co-Authors: Javier M Rodriguez, Ramón García-escudero, María L. Salas, Germán AndrésAbstract:The assembly of African swine fever virus (ASFV) at the cytoplasmic virus factories commences with the formation of precursor membranous structures, which are thought to be collapsed cisternal domains recruited from the surrounding endoplasmic reticulum (ER). This report analyzes the role in virus morphogenesis of the structural Protein P54, a 25-kDa polypeptide encoded by the E183L gene that contains a putative transmembrane domain and localizes at the ER-derived envelope precursors. We show that Protein P54 behaves in vitro and in infected cells as a type I membrane-anchored Protein that forms disulfide-linked homodimers through its unique luminal cysteine. Moreover, P54 is targeted to the ER membranes when it is transiently expressed in transfected cells. Using a lethal conditional recombinant, vE183Li, we also demonstrate that the repression of P54 synthesis arrests virus morphogenesis at a very early stage, even prior to the formation of the precursor membranes. Under restrictive conditions, the virus factories appeared as discrete electron-lucent areas essentially free of viral structures. In contrast, outside the assembly sites, large amounts of aberrant zipper-like structures formed by the unprocessed core polyProteins pp220 and pp62 were produced in close association to ER cisternae. Altogether, these results indicate that the transmembrane structural Protein P54 is critical for the recruitment and transformation of the ER membranes into the precursors of the viral envelope.
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The structural Protein P54 is essential for African swine fever virus viability
Virus research, 1996Co-Authors: Fernando Rodriguez, Javier M Rodriguez, Victoria Ley, Paulino Gómez-puertas, Ramon Güimil García, Josém. EscribanoAbstract:Protein P54, one of the most antigenic structural African swine fever virus (ASFV) Proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the P54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the P54 gene and beta-glucuronidase selection marker under the control of P54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of P54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of P54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the P54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.
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characterization and molecular basis of heterogeneity of the african swine fever virus envelope Protein P54
Journal of Virology, 1994Co-Authors: Fernando Rodriguez, Javier M Rodriguez, Covadonga Alonso, A Eiras, C Alcaraz, Rafael J Yanez, Jose F Rodriguez, Josém. EscribanoAbstract:It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in Protein P54 (C. Alcaraz, A. Brun, F. Ruiz-Gonzalvo, and J. M. Escribano, Virus Res. 23:173-182, 1992). A recombinant bacteriophage expressing a 328-bp fragment of the P54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against P54 Protein. The sequence of this recombinant phage allowed the location of the P54 gene in the EcoRI E fragment of the ASFV genome. Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a Protein of 183 amino acids with a calculated molecular weight of 19,861. This Protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for Protein processing of several ASFV structural Proteins. In addition, two direct tandem repetitions were also found within this open reading frame. Further characterization of the transcription and gene product revealed that the P54 gene is translated from a late mRNA and the Protein is incorporated to the external membrane of the virus particle. A comparison of the nucleotide sequence of the P54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity. However, when P54 genes from viral clones generated by cell culture passage and coding for P54 Proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat. These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in P54, resulting in stepwise modifications in the molecular weight of the Protein. These duplications and deletions of a tandem repeat sequence array within a Protein coding region constitute a novel mechanism of genetic diversification in ASFV.
Fernando Rodriguez - One of the best experts on this subject based on the ideXlab platform.
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the african swine fever virus Proteins P54 and p30 are involved in two distinct steps of virus attachment and both contribute to the antibody mediated protective immune response
Virology, 1998Co-Authors: Paulino Gomezpuertas, Covadonga Alonso, J.m. Oviedo, Fernando Rodriguez, Alejandro Brun, J.m. EscribanoAbstract:Abstract The nature of the initial interactions of African swine fever (ASF) virus with target cells is only partially known, and to date only the ASF virus Protein p12 has been identified as a viral attachment Protein. More recently, antibodies to viral Proteins P54 and p30 have been shown to neutralize the virus, inhibiting virus binding and internalization, respectively. Therefore, we investigated the role of these Proteins in the receptor-mediated ASF virus endocytosis in swine macrophages, the natural host cells. Proteins P54 and p30, released from ASF virus particles after treatment of virions with a nonionic detergent, bound to virus-sensitive alveolar pig macrophages. Binding of these Proteins was found to be specifically inhibited by neutralizing antibodies obtained from a convalescent pig or from pigs immunized with recombinant P54 or p30 Proteins. The baculovirus-expressed Proteins P54 and p30 retained the same biological properties as the viral Proteins, since they also bound specifically to these cells, and their binding was equally inhibited by neutralizing antibodies. Binding of35S-labeled recombinant P54 and p30 Proteins to macrophages was specifically competed by an excess of unlabeled P54 and p30, respectively. However, cross-binding inhibition was not observed, suggesting the existence of two different saturable binding sites for these Proteins in the susceptible cells. In addition, Protein P54 blocked the specific binding of virus particles to the macrophage, while Protein p30 blocked virus internalization. Both Proteins independently prevented virus infection and in a dose-dependent manner, suggesting that binding interactions mediated by both Proteins are necessary to give rise to a productive infection. The relevance of blockade of virus–cell interactions mediated by P54 and p30 in the protective immune response against ASF virus was then investigated. Immunization of pigs with either recombinant P54 or p30 Proteins induced neutralizing antibodies which, as expected, inhibited virus attachment or internalization, respectively. However, immunized pigs were not protected against lethal infection and the disease course was not modified in these animals. In contrast, immunization with a combination of P54 and p30 Proteins simultaneously stimulated both virus neutralizing mechanisms and modified drastically the disease course, rendering a variable degree of protection ranging from a delay in the onset of the disease to complete protection against virus infection. In conclusion, the above results strongly suggest that Proteins P54 and p30 mediate specific interactions between ASF virus and cellular receptors and that simultaneous interference with these two interactions has a complementary effect in antibody-mediated protection.
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The structural Protein P54 is essential for African swine fever virus viability
Virus research, 1996Co-Authors: Fernando Rodriguez, Javier M Rodriguez, Victoria Ley, Paulino Gómez-puertas, Ramon Güimil García, Josém. EscribanoAbstract:Protein P54, one of the most antigenic structural African swine fever virus (ASFV) Proteins, has been localized by immuno-electron microscopy in the replication factories of infected cells, mainly associated with membranes and immature virus particles. Attempts to inactivate the P54 gene from ASFV by targeted insertion of beta-galactosidase selection marker was uniformly unsuccessful, suggesting that this gene is essential for virus viability. To demonstrate that, we inserted in the TK (thymidine kinase) locus of the virus a construction containing a second copy of the P54 gene and beta-glucuronidase selection marker under the control of P54 and p73 promoters, respectively. Virus mutant clones expressing a second copy of P54 and beta-glucuronidase were used to achieve deletion mutants of the original copy of the gene. Virus mutants expressing only the second inserted copy of P54 and the two selection markers mentioned above were successfully obtained. Therefore, we have demonstrated that the P54 gene product plays an essential role in virus growth, characterizing for the first time in ASFV an essential virus gene.
-
highly specific confirmatory western blot test for african swine fever virus antibody detection using the recombinant virus Protein P54
Journal of Virological Methods, 1995Co-Authors: Carlos Alcaraz, Covadonga Alonso, J.m. Oviedo, Fernando Rodriguez, A Eiras, M De Diego, J.m. EscribanoAbstract:A Western blot technique using a recombinant Protein has been developed to confirm positive results obtained in African swine fever (ASF)-specific antibody detection by ELISA. The new confirmatory Western blot is based on the use of Protein P54, one of the most antigenic ASF virus structural Proteins, expressed in Escherichia coli fused to the N-terminus of MS2 polymerase. The recombinant Western blot assay was highly specific and equally sensitive for ASF virus-infected pigs detection as the conventional Western blot, which uses virus-induced Proteins ranging in molecular weight between 23 and 35 kDa. The novel Western blot assay provides a simpler interpretation of the test, eliminates the possibility of false-positive reactions produced by cellular compounds that contaminate the antigen employed in the conventional technique, and avoids the use of live virus in antigen production.
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characterization and molecular basis of heterogeneity of the african swine fever virus envelope Protein P54
Journal of Virology, 1994Co-Authors: Fernando Rodriguez, Javier M Rodriguez, Covadonga Alonso, A Eiras, C Alcaraz, Rafael J Yanez, Jose F Rodriguez, Josém. EscribanoAbstract:It has been reported that the propagation of African swine fever virus (ASFV) in cell culture generates viral subpopulations differing in Protein P54 (C. Alcaraz, A. Brun, F. Ruiz-Gonzalvo, and J. M. Escribano, Virus Res. 23:173-182, 1992). A recombinant bacteriophage expressing a 328-bp fragment of the P54 gene was selected in a lambda phage expression library of ASFV genomic fragments by immunoscreening with antibodies against P54 Protein. The sequence of this recombinant phage allowed the location of the P54 gene in the EcoRI E fragment of the ASFV genome. Nucleotide sequence obtained from this fragment revealed an open reading frame encoding a Protein of 183 amino acids with a calculated molecular weight of 19,861. This Protein contains a transmembrane domain and a Gly-Gly-X motif, a recognition sequence for Protein processing of several ASFV structural Proteins. In addition, two direct tandem repetitions were also found within this open reading frame. Further characterization of the transcription and gene product revealed that the P54 gene is translated from a late mRNA and the Protein is incorporated to the external membrane of the virus particle. A comparison of the nucleotide sequence of the P54 gene carried by two virulent ASFV strains (E70 and E75) with that obtained from virus Ba71V showed 100% similarity. However, when P54 genes from viral clones generated by cell culture passage and coding for P54 Proteins with different electrophoretic mobility were sequenced, they showed changes in the number of copies of a 12-nucleotide sequence repeat. These changes produce alterations in the number of copies of the amino acid sequence Pro-Ala-Ala-Ala present in P54, resulting in stepwise modifications in the molecular weight of the Protein. These duplications and deletions of a tandem repeat sequence array within a Protein coding region constitute a novel mechanism of genetic diversification in ASFV.
Germán Andrés - One of the best experts on this subject based on the ideXlab platform.
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african swine fever virus Protein p17 is essential for the progression of viral membrane precursors toward icosahedral intermediates
Journal of Virology, 2010Co-Authors: Cristina Suarez, Javier Gutierrezberzal, Germán Andrés, Maria Salas, Javier M RodriguezAbstract:The first morphological evidence of African swine fever virus (ASFV) assembly is the appearance of precursor viral membranes, thought to derive from the endoplasmic reticulum, within the assembly sites. We have shown previously that Protein P54, a viral structural integral membrane Protein, is essential for the generation of the viral precursor membranes. In this report, we study the role of Protein p17, an abundant transmembrane Protein localized at the viral internal envelope, in these processes. Using an inducible virus for this Protein, we show that p17 is essential for virus viability and that its repression blocks the proteolytic processing of polyProteins pp220 and pp62. Electron microscopy analyses demonstrate that when the infection occurs under restrictive conditions, viral morphogenesis is blocked at an early stage, immediately posterior to the formation of the viral precursor membranes, indicating that Protein p17 is required to allow their progression toward icosahedral particles. Thus, the absence of this Protein leads to an accumulation of these precursors and to the delocalization of the major components of the capsid and core shell domains. The study of ultrathin serial sections from cells infected with BA71V or the inducible virus under permissive conditions revealed the presence of large helicoidal structures from which immature particles are produced, suggesting that these helicoidal structures represent a previously undetected viral intermediate.
-
African Swine Fever Virus Structural Protein P54 Is Essential for the Recruitment of Envelope Precursors to Assembly Sites
Journal of virology, 2004Co-Authors: Javier M Rodriguez, Ramón García-escudero, María L. Salas, Germán AndrésAbstract:The assembly of African swine fever virus (ASFV) at the cytoplasmic virus factories commences with the formation of precursor membranous structures, which are thought to be collapsed cisternal domains recruited from the surrounding endoplasmic reticulum (ER). This report analyzes the role in virus morphogenesis of the structural Protein P54, a 25-kDa polypeptide encoded by the E183L gene that contains a putative transmembrane domain and localizes at the ER-derived envelope precursors. We show that Protein P54 behaves in vitro and in infected cells as a type I membrane-anchored Protein that forms disulfide-linked homodimers through its unique luminal cysteine. Moreover, P54 is targeted to the ER membranes when it is transiently expressed in transfected cells. Using a lethal conditional recombinant, vE183Li, we also demonstrate that the repression of P54 synthesis arrests virus morphogenesis at a very early stage, even prior to the formation of the precursor membranes. Under restrictive conditions, the virus factories appeared as discrete electron-lucent areas essentially free of viral structures. In contrast, outside the assembly sites, large amounts of aberrant zipper-like structures formed by the unprocessed core polyProteins pp220 and pp62 were produced in close association to ER cisternae. Altogether, these results indicate that the transmembrane structural Protein P54 is critical for the recruitment and transformation of the ER membranes into the precursors of the viral envelope.
